The present invention relates to DNA sequencing and genotyping, and more particularly, to DNA sequencers and genotypers that use optical fluorescence detection techniques.
The basic biological characteristics of a living organism are contained in its genes or genetic code. In humans, for example, a person's biological characteristics are controlled by the genetic code contained in 23 chromosome pairs. Each chromosome contains differing genes.
The specific details of a genetic code are contained in long double helical molecules called deoxyribonucleic acid or DNA. The DNA consists of long sequence pairs of four nucleotides or bases: adenosine, cytosine, guanosine or thymidine, commonly referred to by the letters A, C, G, and T, respectively. In the double helix, the A and T nucleotides are complementary and the C and G nucleotides are complementary. Thus, the DNA molecules consist of two complementary strands that are bound together by the complements.
It is often advantageous to know the sequence of the DNA nucleotides associated with a particular gene. For example, genetic defects can be detected by analyzing an organism's genes. The DNA nucleotides for several bacteria and viruses have been sequenced, and currently, sequencing of the entire human genome is in progress. The entire human DNA consists of approximately 3 billion nucleotides or base pairs.
Existing high speed DNA sequencers use electrophoresis gel techniques, in conjunction with fractioning enzymes and fluorescent tags or markers, to separate residual DNA sequence fragments as they travel through a gel. More specifically, each DNA fragment has an incrementally different molecular weight and size. Because the mobility is related to the fragment's weight, structure, and charge, the differing fragments travel through the gel at differing speeds. Thus, the time it takes a fragment to travel through the gel relates to the fragment's mobility.
Generally, four fluorescent tags are used. These tags bind on the residual fragments in accordance with the exposed end base, if using dye terminator chemistry, or are attached to primers that are used to initiate the sequencing reaction, if using dye primer chemistry. The sequence is read by causing the fluorescent markers to fluoresce. The four fluorescent tags generally are selected to have a strong fluorescence peak that is separated from the strong fluorescence peak of the remaining tags. An optical instrument detects the emitted fluorescence signals.
Existing DNA Sequencers use an optical filter having a pass-band that is centered about the appropriate wavelength to distinguish between the dyes, and thus the fragments. The optical instrument typically includes a simple spectrometer or a filter wheel and a photomultiplier. The filter wheel has several colored filters, each filter passing light within a wavelength band corresponding to the spectral peak of one of the tags. A simple spectrograph has a wavelength-dependent light disperser such as a prism. The light disperser spreads, generally along a line, the different wavelengths of fluorescent light from the DNA fragments traveling in the gel. Four detectors are placed along the spreading line of the spectrograph at differing locations that correspond to the wavelengths associated with the fluorescent tags.
Fluorescent dyes have been found to be good fluorescent tags. Using dye primer chemistry, the tag associated with the C base often is fluorescein-5-isothiocynate (FITC) which has an emission or fluorescence peak at about 525 nanometers. The tag associated with the T base often is Texas Red, which has a fluorescence peak at about 620 nanometers. The tag associated with the G base often is Tetramethyl rhodamine isothiocynate (TRITC), which has a fluorescence peak at about 580 nanometers. Finally, the marker associated with the A base often is 4-fluoro-7nitro-benzofurazan (NBD-fluoride) which has a fluorescence peak at about 540 nanometers. Commercially, four universal primers, respectively labeled with dyes called FAM (C), TAMRA (G), JOE (A), and ROX (T), are available from Applied Biosystems, Inc. (ABI) of Foster City, Calif.
The fluorescent dyes indicated above are subject to bleaching which limits the excitation light's power level and thus the intensity of the emitted fluorescence signal from the dyes. The upper limits on fluorescence intensity limit the signal-to-noise ratio (SNR) and eventually the system's throughput.
Accordingly, there exists a need for a sequencing or genotyping system that has increased throughput and sensitivity over systems using four dyes that are distinguished by their respective fluoresce peaks. The present invention satisfies these needs.